Currently there is considerable interest in adult stem cells from a variety of tissues because of their potential to contribute to our basic understanding of differentiation and development as well as their long-term therapeutic potential. Of all adult tissues, the small intestinal epithelium has by far the most stem cells and the highest rate of turnover. The intestinal stem cells are located deep in the crypts of Leiberkuhn and have been demonstrated to be multipotent precursors of the 4 principal lineages within the epithelium. Although the in vivo behavior (e.g. kinetics) of intestinal stem cells has been studied for many years, our understanding of the properties of these cells has been greatly hampered by lack of methods to isolate, identify and characterize them. Likewise, there is currently a lack of suitable cell transplantation models in which to study proliferation and differentiation of putative intestinal epithelial stem cells. Finally, in light of recent findings in both mice and humans, the possibility that stem cells from other tissues make a quantitatively significant contribution to the intestinal epithelium appears worthy of investigation. Thus, the Specific Aims of this exploratory R21 proposal are: 1) To develop flow cytometry methods for isolation of viable stem cells from the epithelium of mouse small intestine. Specifically, to subject intestinal epithelial cells to novel sorting procedures that have been successfully used with bone marrow cells to identify a fraction which is highly enriched in stem cells; 2) To develop a mouse model with local, limited sterilization of intestinal crypts to create an ideal region for subsequent transplantation of putative epithelial stem cells; 3) To transplant putative intestinal epithelial stem cells into damaged regions of mouse small intestine and subsequently assess the morphology as well as the expression of lineage-specific markers in epithelium derived from the transplanted cells; and 4) To repeat the studies of Specific Aim 3 using bone marrow hematopoietic stem cells. These studies should set the stage for a variety of future investigations regarding both the basic biology and the therapeutic potential of intestinal epithelial stem cells.